CRISPR-Cas9: a new and promising playerin gene therapyLu Xiao-Jie,1 Xue Hui-Ying,2 Ke Zun-Ping,3 Chen Jin-Lian,4,1 Ji Li-Juan5

1Department ofGastroenterology, ShanghaiEast Hospital, Tongji UniversitySchool of Medicine, Shanghai,China2The Reproductive Center,Jiangsu Huai’an Maternity andChildren Hospital, Huai’an,China3Department of Cardiology,The Fifth People’s Hospitalof Shanghai, Fudan University,Shanghai, China4Department ofGastroenterology, ShanghaiSixth People’s Hospital (South),Shanghai Jiaotong UniversitySchool of Medicine, Shanghai,China5Department of Rehabilitation,The Second People’s Hospitalof Huai’an, Huai’an, China

Correspondence toDr Chen Jin-Lian, Departmentof Gastroenterology, ShanghaiSixth People’s Hospital (South),Shanghai Jiaotong UniversitySchool of Medicine, Shanghai,China; wqq_021002@163.com or Ji Li-Juan, Departmentof Rehabilitation, The SecondPeople’s Hospital of Huai’an,Huai’an, China.189@whu.edu.cn

Lu Xiao-Jie and Xue Hui-Yingare co-first authors.

Received 30 December 2014Revised 2 February 2015Accepted 2 February 2015Published Online First24 February 2015

To cite: Xiao-Jie L,Hui-Ying X, Zun-Ping K,et al. J Med Genet2015;52:289–296.

ABSTRACTFirst introduced into mammalian organisms in 2013, theRNA-guided genome editing tool CRISPR-Cas9 (clusteredregularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) offers several advantages overconventional ones, such as simple-to-design, easy-to-useand multiplexing (capable of editing multiple genessimultaneously). Consequently, it has become a cost-effective and convenient tool for various genome editingpurposes including gene therapy studies. In cell lines oranimal models, CRISPR-Cas9 can be applied fortherapeutic purposes in several ways. It can correct thecausal mutations in monogenic disorders and thusrescue the disease phenotypes, which currentlyrepresents the most translatable field in CRISPR-Cas9-mediated gene therapy. CRISPR-Cas9 can also engineerpathogen genome such as HIV for therapeutic purposes,or induce protective or therapeutic mutations in hosttissues. Moreover, CRISPR-Cas9 has shown potentials incancer gene therapy such as deactivating oncogenic virusand inducing oncosuppressor expressions. Herein, wereview the research on CRISPR-mediated gene therapy,discuss its advantages, limitations and possible solutions,and propose directions for future research, with anemphasis on the opportunities and challenges ofCRISPR-Cas9 in cancer gene therapy.

INTRODUCTIONGene therapy involves manipulating DNA or RNAfor human disease treatment or prevention. Thestrategies of gene therapy are diverse, such as recti-fying, replacing or deleting the culprit genes ingenetic diseases, producing disabling mutations inpathogen genomes to combat infectious diseases orinducing therapeutic or protective somatic muta-tions. It is a promising therapy for a wide range ofhuman diseases including hematological diseases,1 2

cancer,3 AIDS,4 diabetes,5 6 heart failure,7 and neu-rodegenerative diseases.8 Up to now, there has beenmore than 2000 gene therapy clinical trials world-wide,9 with a few gene therapy products havingalready been approved by authorities, such asGendicine for head and neck squamous cell carcin-oma in China, and Cerepro for malignant braintumours in Europe.10

Targeted genome editing with programmablenucleases, such as zinc finger nucleases (ZFNs) andtranscription activator-like effector nucleases(TALENs), enables diverse genome manipulationsin a site-specific manner, such as gene activation/inactivation, sequence deletion, element replace-ment and chromosomal rearrangement.11 12 Unlikeprevious gene therapy tools that add or insert anexogenous DNA copy into the target cell nucleusor genome, which may give rise to side effects such

as insertional mutations and non-physical expres-sion of proteins, the programmable nucleases use a‘cut-and-paste’ strategy, that is, remove the defectand install the correct, thus representing an prefer-able tool for gene therapy. Recently, a RNA-guidedgenome editing tool termed CRISPR-Cas9 (clus-tered regularly interspaced short palindromicrepeats/CRISPR-associated nuclease 9) added to thelist of programmable nucleases, offers severaladvantages over its counterparts and shows thera-peutic potentials. Herein, we introduce the basicmechanisms and merits of CRISPR-Cas9 ingenome editing, retrospect studies onCRISPR-mediated gene therapy in cell lines andanimal models, discuss its challenges and possiblesolutions and prospect future directions.

MECHANISMS AND MERITS OF CRISPR-CAS9IN GENOME EDITINGFirst applied in mammalian cells in 2013,13 14

CRISPR-Cas9 genome editing tool is adapted frommicrobial adaptive immune defense system(figure 1A). As shown in figure 1B, the core com-ponents of CRISPR-Cas9 are a nuclease Cas9 com-prising two catalytic active domains RuvC andHNH, and a single guide RNA (sgRNA) that isderived from CRISPR RNA (crRNA) and trans-acting CRISPR RNA.15 On the presence of aprotospacer-adjacent motif (PAM) on the oppositestrand, sgRNA directs Cas9 to the target site bybase-pairing, resulting in Cas9-generated site-specific DNA double-strand breaks (DSBs) that aresubsequently repaired by homologous directedrepair (HDR) if the homologous sequences areavailable or otherwise by non-homologous end-joining (NHEJ).13 14 15 HDR leads to precise genecorrection or replacement whereas NHEJ is errorprone and may induce small insert or delete (indel)mutations (figure 1C). Additionally, Cas9 can bereprogrammed into nickase (nCas9) by inactivatingeither RuvC or HNH,16 or into catalytically inactiveCas9 (termed dead Cas9, or dCas9) by inactivatingboth of them (figure 1D).17 dCas9 can be repur-posed as a site-specific DNA-binding domain trans-porting various functional domains to the targetlocus (figure 1D).17

This RNA-guided genome editing tool providesseveral advantages over conventional protein-guided ones such as ZFN and TALEN. First, withCRISPR-Cas9, to target a new site requires onlythe design of a complementary sgRNA (the nucle-ase Cas9 remains the same in all cases), which ismuch simpler than the de novo synthesis of a bulkyguiding protein as in ZFN- or TALEN-based tools.Second, with multiple sgRNAs that target differentgenomic loci, CRISPR-Cas9 can edit these genomic

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loci in parallel, a property called multiplexing.13 15 However,protein-guided genome editing tools such as TALEN also havesome advantages over CRISPR-Cas9. First, the targets ofCRISPR-Cas9 are restricted by the presence of PAM sequenceand a guanine at the 50 end 11, whereas the only restriction ofTALEN targets is the presence of thymine at the 50 end,18 whichmeans that TALEN can target more genome sites thanCRISPR-Cas9. Second, TALEN are generally reported toproduce less off-target effects in relative to CRISPR-Cas9,19 20

partially by virtue of the heterodimeric construction of FokInuclease in TALEN. And moreover, TALEN can bere-engineered to target and cleave mutant mitochondrial DNA

in patient-derived cells,21 whereas mitochondrial genome-targeting CRISPR-Cas9 remains an area to be explored. Formore details on the comparison among these programmablenucleases, please see review articles such as references 11, 18and 20.

RNA interference (RNAi) technology is also a convenient andwidely used way for the in vivo manipulation of gene expres-sion. However, the effects of RNAi are temporary, non-specificand limited to knocking down transcribed genes. CRISPR-Cas9system, on the contrary, can induce both gain- andloss-of-function mutations, and suffers from less off-targeteffects for that CRISPR-Cas9 derives from prokaryote and

Figure 1 Mechanisms of CRISPR-Cas9-mediated genome editing and epigenome modulation. (A) Representative schematic of CRISPR locus (fromStreptococcus pyogenes). (B) Site-specific DNA cleavage by nuclease Cas9 directed by complementary between a single guide RNA (sgRNA) and thetarget sequence on the presence of a PAM on the opposite strand. sgRNA derives from crRNA and tracrRNA. Wild-type Cas9 possesses twocatalytically active domains termed HNH and RuvC, each of which cleaves a DNA strand. (C) Double-strand breaks generated by Cas9 aresubsequently repaired by homologous directed repair (HDR) if the homologous sequences are available or otherwise by non-homologous end-joining(NHEJ). Generally, HDR leads to precise gene correction or replacement whereas NHEJ is error prone and may induce small insert or delete (indel)mutations. (D) Cas9 can be reprogrammed into nickase (nCas9) by inactivating either RuvC or HNH, or into catalytically inactive Cas9 (termeddCas9) by inactivating both of them. dCas9 can be repurposed as a site-specific DNA-binding domain fused to various effectors. crRNA, CRISPRRNA; tracrRNA, trans-activating crRNA; HA, homologous arm; DS, donor sequence; DSD, double-strand DNA donor; ssODN, single-strandedoligonucleotide DNA; PAM, protospacer-adjacent motif.

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hence has less crosstalk with eukaryotic components than RNAipathway does.

With these merits, CRISPR-Cas9 has become as an easy andversatile tool for various genome editing purposes. For example,CRISPR-Cas9 has been used to recapitulate cancer-associatedgenomic alterations both in vitro and in vivo,22–25 providing aquick and convenient platform for functional investigations ofcancer-related genetic mutations. Recently, a more exiting area,that is, the application potentials of CRISPR-Cas9 in genetherapy, has gradually emerged.

CRISPR-CAS9-MEDIATED GENE THERAPY IN CELL LINESAND ANIMAL MODELSEngineering pathogen DNA to combat infectious diseasesCRISPR-Cas9 is derived from microbial adaptive immunedefense system, and thus possesses inherent advantages in engin-eering and disabling pathogen genomes to treat infectiousdiseases.

In August 2013, just 7 months after the CRISPR-Cas9 genomeediting tool was first introduced into mammalian cells,13 14 Ebinaet al26 reported that CRISPR-Cas9 can mutate long terminalrepeat (LTR) sequence of HIV-1 in vitro, resulting in removal ofthe integrated proviral DNA from the part of the host cells and asignificant drop in virus expression. To our knowledge, this studyis the first one to experimentally explore the gene therapy poten-tials of CRISPR-Cas9 system. Recently, another independentstudy harnessed CRISPR-Cas9 to mutate HIV-1 LTR U3 regionand gained similar results.27

Chronic hepatitis B is one the most common infectious dis-eases worldwide, which can lead to liver cirrhosis and cancer.The persistence of hepatitis B virus (HBV) infection is due tothe intrahepatic existence of HBV covalently closed circularDNAs (cccDNAs), which are hard to clear by present therapeu-tics and serve as a reservoir for HBV reactivation. Recently, astudy by Seeger and Sohn28 showed that in HepG2 cells expres-sing HBV, the introduction of cccDNA-targeting CRISPR-Cas9system resulted in both decreased hepatitis B core antigenexpression and mutated cccDNAs, providing impetus forfurther research on the possibility of CRISPR-Cas9-mediatedcccDNA clearance.

Correcting monogenic disordersMonogenetic disorder is caused by single gene defects.Compared with polygenic diseases such as cancer, monogenicdisorders are more amenable to gene therapies. Currently, thecorrection of monogenic disorders represents the most translat-able field in CRISPR-Cas9-mediated gene therapy.

Gene correction in animal germlineGermline modification is a conventional approach for genetherapy studies in animal models, though it is currently notapplicable to humans.

In a mouse model of Duchenne muscular dystrophy (DMD),an inherited X-linked monogenic disease,CRISPR-Cas9-mediated gene editing in the germline gave riseto genetically mosaic offsprings with 2–100% somatic cells car-rying the corrected version of the culprit gene.29 Intriguingly,the extent of phenotype rescue surpassed the percentage ofgene correction, for example, 17% gene corrections resulted in47–60% rescued muscle cells in one of the experimental mouse,indicating a growth advantage imposed by the gene correction.

Study by Wu et al30 serves as another example. One basepairdeletion in exon 3 of Crygc (crystallin gamma C) gene in mousecauses frameshift mutation and cataract phenotype. In such a

mouse model, germline-manipulation with CRISPR-Cas9 systemwas capable of correcting both the mutant gene and cataractphenotype in part of offsprings. Briefly, plasmids expressingCas9 mRNA and a sgRNA (targeting the defect Crygc gene)were injected into murine zygotes carrying Crygc mutation. Ofthe 22 live offsprings, 10 underwent culprit gene modifications(the editing efficiency is 45.45%) either by HDR (n=4) (figure1C left) or by NHEJ (n=6) (figure 1C right). As expected, all ofthe four offsprings undergoing HDR were free of cataract dueto HDR-mediated gene corrections. Interestingly, however, ofthe six offsprings undergoing NHEJ, two were also free of cata-ract thanks to coincidental frameshift restoration resulting fromNHEJ-induced indel mutations, while the rest four remainedcataract phenotype.30 These results highlighted the importanceof HDR in reliable gene corrections. However, as demonstratedin this study, zygote manipulation can only rescue part of theoffsprings in an unpredictable manner. To gain 100% rescueefficiency, Wu et al31 corrected the culprit gene Crygc in mousespermatogonial stem cells (SSCs) with CRISPR-Cas9, and thenpicked out SSC lines that carried the corrected gene withoutundesired genome perturbations. The pups generated by thesepreselected SSCs were unexceptionally cataract free.

Somatic gene correction in adult animalsCRISPR-Cas9 technology can also mediate somatic gene correc-tions in adult animals, bypassing embryo manipulations. Yinet al,32 for example, delivered CRISPR-Cas9 agents and a hom-ologous donor template (in order to increase HDR rate) intoadult mice with hereditary tyrosinemia via tail-vein hydro-dynamic injection, resulting in gene corrections in 0.25% ofliver cells initially, and in 33.5% of liver cells 33 days postinjec-tion (possibly due to selective advantages imposed by the cor-rection), which was sufficient to rescue the disease phenotype.This method is more translatable to human therapeutics becauseit does not involve embryo manipulations.

Gene correction in human stem cells or inducedpluripotent stem cellsBesides in animal models, CRISPR-Cas9 can also exert thera-peutic functions in human stem cells or induced pluripotentstem cells (iPSCs). In human intestinal stem cells collected frompatients with cystic fibrosis, the culprit gene cystic fibrosis trans-membrane conductance regulator was rectified by homologousrecombination during CRISPR-Cas9 genome editing while thepluripotency was retained as demonstrated by formations oforgan-like expansions in cell culture.33 The clinical translatabil-ity of this study gets support from previous study34 in which exvivo cultured colon organoids were successfully transplantedinto mice colons and formed functionally and histologicallynormal crypts.

Non-multipotent somatic cells can be reprogrammed intoiPSCs, with multipotentiality and self-renewal ability similar toembryonic stem cells. iPSC represents an ideal cell tool for genetherapy because patient-derived iPSC can be genetically modi-fied in vitro, then differentiates into desired cells for therapeuticautologous transplantation. With CRISPR-Cas9, gene correc-tions have been successfully conducted in iPSCs derived fromβ-thalassemia patients35 and DMD patients,36 paving the wayfor ultimate clinical applications of gene therapies for these twoinherited diseases based on CRISPR-Cas9 and iPSC.

The above-mentioned monogenic disorders are all caused byloss-of-functions mutations. In monogenic disorders that areresulted from gene duplications (eg, Friedreich’s ataxia), or inautosomal dominant disorders in which the affected genes are

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haplosufficient, CRISPR-Cas9 also holds therapeutic potentialgiven its ability in gene deletion23 and inactivation.24 25

Inducing therapeutic or protective mutationsIn the treatment of non-genetic or polyetiological diseases,CRISPR-Cas9 is also a potential participant by inducing thera-peutic or protective mutations in somatic tissues. For example,mutation in CCR5 (CC chemokine receptor 5) gene that leadsto cell-resistance to HIV-1 infection was achieved in iPSCs withCRISPR-Cas9.37 Interestingly, a recent clinical trial38 showedthat autologous reintroduction of CD4T cells whose CCR5 genewas inactivated in vitro by ZFN was safe and lead to decreasedviral load, lending support to the potential of CRISPR-Cas9 inAIDS gene therapy.

Another example involves the PCSK9 (proprotein convertasesubtilisin/kexin type 9) gene, whose spontaneousloss-of-function mutations in a small fraction of human beingsare associated with lower plasma low-density lipoprotein (LDL)level39 In mice liver, CRISPR-Cas9 was reported to successfullyinduce loss-of-function mutation at PCSK9 gene locus, leadingto elevated hepatic LDL receptor level and dropped plasmacholesterol level, thus holding therapeutic potentials for hyper-cholesterolaemia, a common disorder in modern society.40

CRISPR-mediated cancer gene therapyAlthough only very few gene therapy products were licensed forcancer treatment during the past decades,3 gene therapy remainsone of the hottest spots in cancer research nowadays, given thatcancer is a genetic disease and that gene therapy holds thepromise of combating cancer from inside with minimum sideeffects. Of the more than 2000 gene therapy clinical trialsworldwide, 64.1% were cancer gene therapy.9

Inactivation or clearance of oncogenic virusMany viral infections are associated with carcinogenesis, such asHBV and hepatitis C virus in liver cancer, Epstein-Barr virus(EBV) in nasopharyngeal carcinoma and human papillomavirus(HPV) in cervical cancer. Clearance or inactivation of theseoncogenic viruses can interrupt and may even reverse tumori-genesis process. CRISPR-Cas9 editing system is derived frombacterial native immune system and hence has inherent advan-tage in defense against or clearance of viral infection. Exampleshave already existed of CRISPR-Cas9-mediated antivirus andantiproliferation effects in HPV-positive cervical carcinoma cellline41 and EBV-positive Burkitt’s lymphoma cell line42 Zhenet al41 reported that the transfection of Cas9 and sgRNAs tar-geting HPV16 E6 or E7 into HPV-16-positive cervical carcin-oma cell lines SiHa resulted in the inhibition of cancer cellproliferation both in vitro and in xenograft mouse model.Similarly, in a Burkitt’s lymphoma cell line with latent EBVinfection, CRISPR-Cas9 treatment inhibited cell proliferationand reduced viral load.42 Therefore, CRISPR-Cas9-mediatedinactivation or clearance of oncogenic virus provides a promis-ing and cost-effective option for the prevention and treatmentof virus-associated cancers.

Manipulating cancer genome or epigenome for therapeuticpurposesGiven that cancer is a genetic disease and that CRISPR-Cas9 is aversatile genomic editing tool, it is easy for us to conceive thatto correct oncogenic genome aberrations via CRISPR-Cas9 mayrepresent a promising anticancer strategy. CRISPR-Cas9 cancorrect genetic mutations as demonstrated in monogenic dis-eases,30–36 and modulate epigenetic states.16 43–46 As two sides

of one coin, genetic mutations and epigenetic alterations interactwith each other and cooperate to drive cancer initiation andprogression. Catalytically inactivated dCas9, for example, canbind to DNA elements and suppress their transcriptional activ-ities, a process called CRISPRi.43 44 Moreover, dCas9 can beharnessed as a site-specific DNA binding domain and fused toepigenetic modifiers. Under the guidance of sgRNAs, dCas9 andthe modifiers get access to the target sites and exert epigeneticregulations,16 45 46 a process termed epigenome editing, whichis also a potential tool for anticancer modalities. ThisdCas9-effector strategy has already been applied in genomewide screening study in which gain-of-function mutations inmelanoma cells that confer drug resistance were identified.46

An example of therapeutic genome manipulations comesfrom study by Liu et al47 in which CRISPR-Cas9-mediatedbladder cancer cell-specific genome editing using an AND logicgate (figure 2). It is called ‘AND’ because there are two inputsand a single output, and only the coexistence of the two inputscan generate an output. In this study, the expression of sgRNAsand Cas9 were controlled by two promoters (two inputs), thecancer-specific human telomerase reverse transcriptase promoterand urothelium-specific human uroplakin II promoter (figure2A), respectively. The fact that only in bladder cancer cells existboth the two promoters enables bladder cancer cell-specificexpression of both Cas9 and sgRNAs (figure 2B, left). ThesgRNAs were designed to target LacI gene, which expresses LacIprotein that binds to Lac operator and hence suppresses theexpression of the downstream effector gene (the output) (figure2C, left). In bladder cancer cells, Cas9 and sgRNAs were bothexpressed, mutating LacI gene and abrogating its suppression onthe effector gene, leading to bladder cancer cell-specific expres-sion of the effector (figure 2C, right). In this study, the effectorgenes were p21, E-cadherin and Bax (BCL2-associated X),leading to cancer-specific growth inhibition, migration suppres-sion and apoptosis, respectively. Theoretically, the effector canbe any oncosuppressors (figure 2C) or oncogenes (figure 2D),or suicide genes that cause tumour inhibition or destruction.

However, CRISPR-Cas9-mediated (epi)genetic modulationsfor cancer therapy suffer from several limitations. First, canceris a polygenic and heterogeneous disease. Genomic aberrationprofiles are different in tumours among patients and in tumoursduring different stages or from different sites within a patient,which renders (epi)genome manipulation therapy in cancer ahighly tailored and dynamic process relying on spatiotemporalcontents. In current conditions, it is a mission hard to accom-plish, even with the assistance of CRISPR-Cas9 system. Second,unlike in monogenic diseases that can be rescued at an initialgene correction efficiency as low as 0.25%,32 effective (epi)genome manipulation therapy in cancer requires fairly high editefficiency because the unedited cells retain unreduced malig-nancy, possess selective advantages over the ‘corrected’ cells andproliferate quickly, rendering gene therapy ineffective shortly.However, such a high edit efficiency is still beyond the reach ofcurrent CRISPR-Cas9 technology.30 32

CHALLENGES AND POSSIBLE SOLUTIONSOff-target effectsBecause CRISPR-Cas9 causes permanent genome alterations, itsoff-target effects must be accurately profiled and controlledwhen applied in gene therapy. Current off-target identificationmethods comprises mainly of silicon prediction and in vitroselection, which are based on the complementarity betweensgRNAs and potential off-target sequences. However, siliconmethods can only identify part of the off-target cleavage.48 And

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moreover, DNA binding and cleavage by Cas9 are in some casesuncoupled, that is, Cas9 can bind to but not cleave DNAsequences that are partially complementary to sgRNA49 andexert epigenomic regulatory effects,43 44 which is unpredictableby silicon methods relying on base pairing. So, more unbiasedmethods are needed to provide a comprehensive off-targetprofile, such as genome-wide characterisation of Cas9 bindingprofile by chromatin immunoprecipitation sequencing analysis,49

and genome-wide identification of Cas9 cleavage profile byGUIDE-seq.48

Several methods have been developed to reduce the off-targeteffects. First, both the structure and composition of the guideRNA can affect the frequency of off-target effects.50 To select atarget site that has no homologous sequence throughout the

genome is a practical way for reducing off-target effects.Second, the using of a pair of Cas9 nickases (a mutant form ofCas9 that generates single-stranded break rather than DSB) togenerate paired nicks on the two strands of target sequence cansignificantly increase target specificity because off-target singlenicks are faithfully repaired.51 It is estimated that this doublenicks strategy can increase target-site specificity by about 1500times.15 Third, sgRNA truncated by 2–3 nt are reported to reducethe off-target effects possibly because shorter sgRNA sequence hasa decreased mismatch tolerance.52 Fourth, delivery vehicles bywhich Cas9 and sgRNAs enter into cells can also affect on- to off-target activity ratio. Cell-penetrate-peptide-mediated delivery wasreported to achieve higher on- to off-target activity ratio comparedwith plasmid-mediated delivery.53 And last, the combination of

Figure 2 CRISPR-Cas9-mediated gene (in)activation in cancer cells. (A) The expression of single guide RNAs (sgRNAs) and Cas9 are controlled bycancer-specific promoter and tissue-specific promoter, respectively. Consequently, only in the specific cancer cells can express both of them. (B) ThesgRNAs are designed to target LacI gene or oncogene. (C) In normal cells, LacI protein expressed by LacI gene binds to Lac operator and suppressesthe expression of the downstream effector gene. In specific cancer cells, Cas9 and sgRNAs are both expressed, mutating LacI gene and abrogatingits suppression on the effector gene, leading to its cancer-specific expression. (D) Loss-of-function mutation of oncogene in cancer cells induced byCRISPR-Cas9 editing. (This figure is partially based on the study by Liu et al47).

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CRISPR-Cas9 with other nuclease may also help. Tsai et al54

reported that fusing FokI nuclease to dCas9 generates a dimericRNA-guided FokI nuclease that has an improved specificity com-pared with wild-type CRISPR-Cas system.

Editing efficiencyDelivery and editing efficiency has long been a crux of genetherapy applications,4 55 56 especially for cancer. As statedabove, cancer gene therapy necessitates high editing efficiency,which is still beyond the reach of current CRISPR-Cas9 tech-nologies. The solutions of this problem lie in future the devel-opment of more efficient delivery vectors, more powerfulsgRNA, and more potent Cas9. Adeno-associated virus (AAV)vectors are commonly used gene delivery tools for gene therapyresearch and clinical trials because of their efficiency andsafety.1 57 58 However, the commonly used Cas9 gene derivedfrom Streptococcus pyogenes is too big to transduce with wild-type AAV due to its limited genetic cargo. Using smaller Cas9orthologs59 derived from other microbes or engineering AAV toincrease its genetic cargo57 may represent feasible ways toenhance delivery efficiency. Though cell-penetrating peptide-mediated delivery of CRISPR-Cas9 into cells was reported toincrease editing efficiency, the optimal editing efficiency was stillunder 20% in vitro.59 The delivery of purified recombinantCas9 protein instead of Cas9 gene into human cell line, on theother hand, can achieve an editing efficiency as high as 79%.60

HDR rateThe DSBs produced by CRISPR-Cas9 are subsequently pro-cessed by NHEJ or HDR. In many cases, HDR results indesired gene modifications while NHEJ gives rise to undesiredindels that cause uncontrollable gene disruptions. As shown inthe study by Wu et al30 among mice genetically edited byCRISPR-Cas9, all HDR-mediated editing corrected the causativegene defects and rescued disease phenotype while only a smallpart of NHEJ-mediated editing had the corrective effect.Therefore, increasing HDR rate can improve the efficiency andreliability of CRISPR-Cas9-mediated gene therapy whiledecrease genomic toxic effects concurrently.

Cell cycle phase dominates the choice between NHEJ andHDR, with HDR taking place mainly in S and G2 phases due tothe availability of sister chromatids as templates.61 Nature of thedonor DNA also influences HDR rate.61 62 Via cell cycle syn-chronisation techniques, Lin and colleagues63 conducted timeddelivery of Cas9 protein and sgRNA complexes into humancells and gained HDR rate up to 38%. The authors also demon-strated that longer homologous arm and single-stranded oligo-nucleotide DNA template (compared with double-strand DNAtemplates) can increase HDR rate. Delivery vector is anotherinfluential factor of HDR rate. In a study by Holkers et al,64

CRISPR-Cas9 and DNA donor delivered via protein-cappedadenoviral vector resulted in higher HDR rate versus viaintegrase-defective lentiviral vector or non-viral vector tem-plates. Moreover, previous study showed that bacteriumDeinococcus radiodurans can repair its genome form hundredsof short DNA fragments resulting from exogenous damages.This highly efficient DNA repair systems in microbes might beexplored and harnessed in the future to increase HDR rate ineukaryote systems.65

PERSPECTIVES AND CONCLUSIONCompared with monogenetic diseases, studies onCRISPR-mediated gene therapy in polygenic and polyetiologicdiseases lag behind. To prospect future applications of

CRISPR-Cas9 technology in gene therapy for all human diseasesis infeasible in a single review. Herein, we select cancer as anexample to discuss future directions.

As stated above, CRISPR-Cas9 based (epi)genome manipula-tion therapies in cancer are hampered by heterogeneity in onco-genic mutation profiles and the requirement of pretty highediting efficiency. Inactivation or clearance of oncogenic viruswith CRISPR-Cas9 is a feasible option but this strategy islimited to virus-associated cancer types and has limited thera-peutic efficacy because on initiation, cancer can progress, at leastpartially, independently of the underlying viral infection. On theother hand, oncolytic virotherapy or host genome modificationmay represent more promising modalities.

Some virus can be genetically engineered to specifically infectand replicate in cancer cells, killing them through virus-mediated cytotoxicity or enhanced anticancer immune response.Oncolytic virotherapy is one of the most promising fields incancer gene therapy. Many oncolytic viruses have been tested inclinical trials,66 with one of them licensed in China.67

CRISPR-Cas9 can play a part in oncolytic virotherapy in severalways, such as adding a cancer-specific promoter to genes thatare indispensable for viral replication, or inducing mutations inviral genome whose defects can be complemented by cancer-specific metabolites.

Currently, another hot spot in cancer gene therapy is themodifications of the host cells to enhance anticancer immuneresponses,68 or to increase resistance to chemo- and radiotoxi-city.3 Adoptive T-cell therapy, for example, in which geneticallyengineered T cells were re-infused back into patients, has shownsurvival improving effects in clinical trials.69 Given thatCRISPR-Cas9 has been applied in T cell modification70 andimmune modulation,70 71 it is reasonable for us to envision itsapplication in cancer immune therapies.

In conclusion, the RNA-guided genome editing toolCRISPR-Cas9 offers several advantages over protein guidedcounterparts and RNAi techniques. It has shown therapeuticpotentials in cell lines or animal models for infectious diseases,monogenic diseases and cancer. Before it is translatable to theclinic to benefit patients, several challenges must be overcome,such as ways to reliably profile and control the off-target effects,ways to increase editing efficiency and ways to improve HDRrate. However, with the rapid advance in CRISPR-Cas9 technol-ogy, we can still optimistically anticipate that, in the future, itmay revolutionise gene therapy research and become a conveni-ent and versatile tool for human gene therapy practice.

Correction notice This article has been corrected since it published Online First.The fifth sentence in the Editing efficiency section has been corrected to read‘Using smaller Cas9 orthologs...’

Contributors CJ-L and JL-J conceived the idea and collected literatures; LX-J andKZ-P read through and analysed literatures; LX-J wrote the manuscript while XH-Yand JL-J revised it.

Funding National High Technology Research (863) Project of China(2012AA020204).

Competing interests None.

Provenance and peer review Commissioned; externally peer reviewed.

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